An actinomycete, Streptomyces species, has been widely utilized all over the world as a producer for various useful substances including pharmaceuticals and pharmaceutical intermediates as typified by an antibiotic or an immunosuppressor, and is industrially very useful microorganisms. Even with development of genetic engineering technologies in recent years, a classical method by mutagen treatment is still predominant in breeding a high producer strain for useful substances in the actinomycete including the Streptomyces species because not only homologous recombination on the chromosome but also gene transfer and transformation as a vector are currently limited to certain actinomycete strains.
A genome sequence has been determined also in the actinomycete, Streptomyces species, in recent years. Therefore, researches are expected to be actively conducted on new physiologically active substances based on genome information in the future. However, for the purpose of developing such substances, linking of genetic information in the genome with production of the useful substances or an enzyme protein that catalyzes synthesis thereof is important, and a host-vector system applicable as a tool is essential.
A gene expression system in microorganisms such as Escherichia coli and yeast has been widely spread and commonly utilized. However, proteins that can be expressed as an active form in a common expression system therefor is limited. Similarly, even though an industrially important gene (P-450 or the like) from the actinomycete can be expressed in closely-related Streptomyces species, the gene can not be expressed as an active protein or enzyme in the common system therefor in many cases.
Several gene expression systems in the Streptomyces species have been developed. In one case, a powerful gene expression system in which large quantities of recombinant proteins as high as 40% of all intracellular soluble proteins can be produced has been reported (Non-patent document 1: Herai et al., Proc Natl Acad Sci USA. 2004 Sep. 28; 101(39): 14031-5. Epub 2004 Sep. 17). However, the systems are provided for the purpose of producing the recombinant proteins. An actinomycete host-vector system that can be applied for producing useful compounds by recombinant microorganisms is virtually unknown. The reason therefor is that achievement of high productivity of a secondary metabolite by using the recombinant microorganisms simultaneously requires specific inducible gene expression during a period suitable for producing the secondary metabolite definitive to the host and also an improvement of precursor supply ability of the host.
Meanwhile, among reactions catalyzed by an organism and a biocatalyst (enzyme) in the organism, reactions catalyzed by ATP-dependent enzymes include many physiologically and also industrially useful reactions. However, ATP is very expensive. Therefore, use of a remarkable amount of ATP as a raw material is quite difficult in industrial production. Thus, many ATP regeneration systems have been reported in which ATP once consumed in the reaction is regenerated by utilizing other energy substances (Non-patent document 2: Zhao & von der Donk, Curr Opin Biotechnol. 2003 December; 14(6): 583-9).
An example has been recently reported in which a thermostable polyphosphate kinase is coexpressed together with an ATP-dependent D-alanine-D-alanine ligase as a dipeptide synthetase in Escherichia coli to carry out dipeptide synthesis reaction using the engineered cells, and thus dipeptide synthesis has been achieved with the aid of ATP regeneration system mediated by the polyphosphate kinase. (Non-patent document 3: Sato et al., J Biosci Bioeng. 2007 February; 103(2): 179-84). In the method, the dipeptide synthesis has been achieved at a yield of 80% (mol/mol) as an added D-alanine without adding ATP from outside a system. However, the amount of the product was not so high with a productivity of approximately 0.02 mol/l, indicating that the amount of ATP available for the reaction was approximately 0.02 mol/l.
Several Streptomyces species, a certain Kitasatospora strain, and a Epichloe strain, a filamentous fungi have been reported to extracellularly perform secretory production of ε-poly-L-lysine that is widely utilized as a food preservative in Japan, Republic of Korea and the United States (Non-patent document 4: Nishikawa & Ogawa, Appl Environ Microbiol. 2002 July; 68(7): 3575-81). Among such e-poly-L-lysine producers, S. albulus NBRC14147 is particularly industrially useful because the S. albulus NBRC14147 shows a remarkably high productivity.
In recent years, an ε-poly-L-lysine synthetase (Pls) and a gene thereof (pls gene) in S. albulus have been identified, and ε-poly-L-lysine has been elucidated to be directly synthesized from L-lysine and ATP both as a precursor (Non-patent document 5: Yamanaka et al., Nat Chem Biol. 2008 December; 4(12): 766-72. Epub 2008 Nov. 9, Patent document 1: JP 2008-263868 A). Moreover, a ε-poly-L-lysine high producer mutant has been derived from S. albulus wild strain by a mutagen treatment (Non-patent document 6: Hiraki et al., Seibutsu-kogaku Kaishi 76(12) pp. 487-493 1998 12 25; Non-patent document 7: Kahar et al., J Biosci Bioeng. 2001; 91(2): 190-4), and has been actually utilized for industrial production of the ε-poly-L-lysine.